Electronically-indexed solid-phase assay for biomolecules

ABSTRACT

Disclosed are materials and methods for detecting biomolecules in samples employing transponders having memory elements associated with particle(s) used as a solid phase in art assay, and information pertinent to the assay is encoded on the transponder memory elements. A dedicated read/write device is used remotely to encode or remotely to read the information encoded on the transponder memory elements. The invention can be used in direct or competitive ELISA-type assays, or in multiplex assays for the simultaneous assay of several analytes.

BACKGROUND OF THE INVENTION

This invention relates to materials and methods for detectingbiomolecules in samples, and more particularly to a particulate solidphase having for encoding information concerning the assay, and toassays employing such a solid phase.

Solid phase assays have been used to determine the presence and/or theconcentration of biomolecules, such as proteins, peptides, nucleicacids, carbohydrates and lipids. Solid-phase assays can be performed ina variety of fluids, e.g., simple buffers, biological fluids, such asblood, serum, plasma, saliva, urine, tissue homogenates, and manyothers.

In solid phase assays, small beads, or microparticles, are typicallyused as the solid phase to capture the analyte. Solid phasemicroparticles can be made of a variety of materials, such as glass,plastic or latex, depending on the particular application. Some solidphase particles are made of ferromagnetic materials to facilitate theirseparation from complex suspensions or mixtures.

In conventional solid-phase assays, the solid phase mainly aids inseparating biomolecules that bind to the solid phase from molecules thatdo not bind to the solid phase. Separation can be facilitated bygravity, centrifugation, filtration, magnetism, immobilization ofmolecules onto the surface of the vessel, etc. The separation may beperformed either in a single step in the assay or, more often, inmultiple steps.

Often, it is desirable to perform two or more different assays on thesame sample, in a single vessel and at about the same time. Such assaysare known in the art as multiplex assays. Multiplex assays are performedto determine simultaneously the presence or concentration of more thanone molecule in the sample being analyzed, or alternatively, to evaluateseveral characteristics of a single molecule, such as, the presence ofseveral epitopes on a single protein molecule.

One problem with conventional multiplex assays is that they typicallycannot detect more than about five analytes simultaneously, because ofdifficulties with simultaneous detection and differentiation of morethan about five analytes. In other words, the number of differentanalytes that may be assayed simultaneously is limited by the solidphase.

SUMMARY OF THE INVENTION

This invention overcomes many of these problems by the use oftransponders associated with the solid phase beads to index theparticles constituting the solid phase. Thus, each individualtransponder-containing solid phase particle can be assigned a uniqueindex number, electronically encoded inside the particle, that can beretrieved by the scanner device at any time, e.g., at one time duringthe assay, at multiple times during the assay, or continuously duringthe assay. The index number may relate to the time and date on which theassay was performed, the patient's name, a code identifying the type ofthe assay, catalog numbers of reagents used in the assay, or datadescribing the progress of the assay, such as temperature duringdifferent steps of the assay.

In an electronically-indexed multiplex assay of this invention, two ormore transponders, each encoded with a different index number andconstructed to bind a different analyte, are incubated with the samplein a single vessel. After necessary additions, incubations and washesare performed, which are similar to incubations and washes in existingassays, the solid phase is analyzed to detect a label indicative ofbinding of the analyte to the solid phase, such as fluorescence, color,radioactivity or the like. Solid phase analysis is either preceded orfollowed by the decoding of the index number on the transponder.

Determination of the label and decoding of the memory of the transpondercan be done manually on two different instruments, such as a fluorometerand a dedicated scanner, although a single automated instrument thatwould perform both functions may be used. Such an instrument can be amodified fluorometer in which the scanner is mounted in the proximity ofthe fluorometer readout window, and reading the sample fluorescence anddecoding the transponder are coordinated by a central computer. Inaddition, such an instrument can be equipped with an automated transportsystem for transponders.

In one aspect, the present invention provides an electronically-indexedsolid phase particle for use in solid phase assays for biomolecules,comprising a transponder and a member of a biomolecular binding pairattached to the transponder.

In another aspect, the present invention provides a method of detectingbiomolecules in a sample using solid phase particles havingtransponders.

In another aspect, the present invention includes a kit for detectingbiomolecules in a sample using transponders, comprising assay vessels, aprobe reagent, and a labelled conjugate reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a simple assay of thisinvention.

FIG. 2 is a schematic representation of a multiplex assay of thisinvention.

FIG. 3 is a cross-sectional view of a solid phase particle with atransponder and a primary layer of biomolecules bound to a surfacethereof.

FIG. 4 is a schematic diagram of the signal pathway for encoding anddecoding data on the transponders.

FIG. 5 is a schematic representation of a miniature transponder.

FIG. 6 is a plan view of a miniature transponder.

FIG. 7 is a plan view of a transport system/analytical instrument forimplementing the present invention.

FIG. 8 is a plan view of a modified flow cytometer for high speedanalysis of solid phase particles of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 depicts a simple assay of the invention. A solid phase particle10, with a transponder 12 is derivatized by attaching an antibody 11 tothe outer surface 16 of the particle 10. Information concerning theassay, e.g., the assay lot number, is encoded on the transponder, eitherby the manufacturer of the transponder, or by the user with a remoteread/write scanner device (not shown). The derivatized particle 10 isincubated with a sample. Antigert 13 present in the sample is bound bythe antibody 11 attached to the particle 10. A second,fluorescent-labelled antibody 15 that binds to the antigen 13 is addedto the sample mixture, and the particle 10 is thoroughly washed toremove unbound components. The labelled antibody 15 is detected with afluorometer to identify those transponders 12 that have antigen 13 boundthereto, and the transponder 12 is decoded using the scanner device (notshown) to retrieve the information encoded thereon. The detection anddecoding steps may be done separately or may be done simultaneously.Alternatively, the beads may be pooled into a vessel in no particularorder with mixing allowed, and passed through a reader (not shown) thatdetermines and records the fluorescence and, at the same time, decodesthe lot number recorded in the transponder 12. It is important to notethat when encoding or reading data on a transponder, other transpondersmust be shielded by a metal barrier, or other means to prevent theelectromagnetic radiation from reaching transponders other than thespecific target.

A multiplex assay according to this invention is conducted in a similarmanner, as depicted in FIG. 2, with two or more transponders 12 in eachassay vessel (not shown) to detect more than one analyte simultaneously.The transponders 12 are divided into two or more classes 12 and 12',each class having a distinct index number identifying the class, andeach class having different antibody 11 and 11' bound to the surface 16of the particle 10 and 10'. Each class of transponder 12, 12' isseparately encoded, either by the manufacturer or by the user with aread/write scanner device (not shown), with an index number to identify,e.g., the antibody 11 bound to the surface 16 of the particle 10. Again,it is necessary to shield other, non-target transponders during theencoding process. The transponders 12, 12' are incubated in the samplevessel and antigen 13, 13' binds to the respective antibody 11, 11'.Second fluorescent-labelled antibodies 15, 15' that bind to the antigens13, 13' are added to the sample vessel to bind to the antigens 13, 13'.The transponders 12, 12' are then washed thoroughly to remove unboundsample components and reagents. The labelled antibody 15, 15' isdetected with a fluorometer to identify those transponders 12, 12' thathave antigen 13, 13' bound thereto, and the transponders 12, 12' aredecoded using the scanner device (not shown) to retrieve the informationencoded thereon. The detection and decoding steps may be done separatelyor may be done simultaneously. Alternatively, the particles 10, 10' maybe pooled into a vessel in no particular order with mixing allowed, andpassed through a reader (not shown) that determines and records thefluorescence and, at the same time, decodes the index number recorded inthe transponder 12, 12'.

The assays of the present invention may be used with a variety ofanalytes, including covalently modified proteins and peptides, proteinor peptide conjugates, small molecules, ribonucleic acid (RNA), modifiednucleic acids and analogs of nucleic acids (in particularprotein-nucleic acids, PNAs). The analyte may be a complex ofbiomolecules, such as a virus particle, a protein-nucleic acid complex,or a protein-hapten complex. The analyte may be a cell, and in such casethe relevant molecules that participate in the binding process duringthe assay are typically cell surface receptors or other elements of thecell wall or membrane. Likewise, the sample may be presented in avariety of forms, such as a solution in a simple buffer, or a complexbiological fluid, such as blood, serum, urine, saliva, and many others,or it can be mixed with many other analytes which are simultaneouslybeing assayed for in the multiplex format.

The biomolecules deposited as a primary layer on the surface of thetransponder may take a variety of forms, as well, such as covalentlymodified proteins and peptides, protein or peptide conjugates, smallmolecules (haptens), ribonucleic acid (RNA), modified nucleic acids andanalogs of nucleic acids (in particular protein-nucleic acids, PNAs).The biomolecules can be made in vivo, or in an enzymatic reaction invitro, or chemically synthesized, either directly or throughcombinatorial synthesis, or may be a fragment of any of the aboveproducts. The purity of the biomolecules deposited as a primary layer onthe surface of the transponder can vary as well, from unpurified,partially purified to pure compounds. The biomolecules, their complexesand aggregates, including subcellular structures or cells, can bedeposited as a primary layer on the surface of the transponder by avariety of means including, for example, chemical conjugation to anactive group on the support, direct chemical synthesis, adhesion ornon-specific binding through hydrophobic interactions.

FIG. 3 depicts a solid phase particle 10 for use in the presentinventive methods. The solid phase particle 10 comprises a glass beadwith a transponder 12 associated with it, and a member of a biomolecularbinding pair (e.g., an antibody or an antigen) attached to the surface16 of the particle 10 as a primary layer 14. The glass surface 16 of thebeads is derivatized through aminoalkylsilane treatment and addition ofa cross-linker, to provide primary amine groups on a solid support forfurther derivatization. The transponder 12 is equipped with a memoryelement.

A transponder is a radio transmitter-receiver activated for transmissionof data by reception of a predetermined signal and may also be referredto as a microtransponder, a radio transponder, a radio tag, etc. Thesignal comes from a dedicated scanner that also receives and processesthe data sent by the transponder in response to the signal. The scannerfunction can be combined with the write function, i.e., the process ofencoding the data on the transponder. Such a combination instrument isreferred to as a scanner read/write device. An advantage of thetransponder-scanner system is that the two units are not electricallyconnected by wire, but are coupled inductively, i.e., by the use ofelectromagnetic radiation, typically in the range from 5-1,000 Khz, butalso up to 1 GHz and higher.

FIG. 4 is a flow chart illustrating the communication between thetransponder 12 and a remote scanner read/write device 18. Thetransponder 12 is encoded with data sent by electromagnetic waves from aremote scanner read/write device 18, unless the transponder 12 waspre-encoded by the manufacturer. After the assay steps are completed,the beads 10 are analyzed to detect the presence of a label indicativeof binding of analyte and the transponders 12 are decoded. The scanner18 sends a signal to the transponder 12. In response to the signal, thetransponder 12 transmits the encoded data to the scanner 18.

Some transponders similar to the type employed in the present inventionare available commercially. For example, BioMedic Data Systems Inc.(BMDS, 255 West Spring Valley Ave., Maywood, N.J.) manufactures aprogrammable transponder for use in laboratory animal identification.The transponder is implanted in the body of an animal, such as a mouse.The transponder is glass-encapsulated to protect the electronics insidethe transponder from the environment. One of the types of transpondersmanufactured by this corporation, model IPTT-100, has dimensions of14×2.2×2.2 mm and weighs 120 mg. The transponder is user-programmablewith up to 16 alphanumeric characters, the 16th letter programmableindependently of the other 15 letters. It has a built-in temperaturesensor as well. The electronic animal monitoring system (ELAMS) includesalso a scanner read/write system, such as the DAS-5001 console system,to encode or read data on/from the transponder. The construction of thetransponder and scanner is described in U.S. Pat. Nos. 5,250,944,5,252,962, and 5,262,772, the disclosures of which are incorporatedherein by reference. Other similar transponder-scanner systems includemulti-memory electronic identification tag (U.S. Pat. No. 5,257,011) byAVID Corporation (Norco, Calif.) and a system made by TEMIC-Telefunken(Eching, Germany). AVID's transponder has dimensions of 1 mm×1 mm×11 mm,and can encode 96 bits of information.

The commercially-available transponders are relatively large in size.The speed at which the transponders may be decoded is limited by thecarrier frequency and the method of transmitting the data. In typicalsignal transmission schemes, the data are encoded by modulating eitherthe amplitude, frequency or phase of the carrier. Depending on themodulation method chosen, compression schemes, transmission environment,noise and other factors, the rate of the signal transmission is withintwo orders of magnitude of the carrier frequency. For example, a carrierfrequency of 1,000 Hz corresponds to rates of 10 to 100,000 bits persecond (bps). At the rate of 10,000 bps the transmission of 100 bitswill take 0.01 sec. The carrier frequency can be several orders ofmagnitude higher than 1,000 Hz, so the transmission rates can beproportionally higher as well.

Therefore, the limiting factor in the screening process is the speed atwhich the transport mechanism carries the transponders through the readwindow of the fluorometer/scanner device. In state-of-the-art flowcytometers, the rate of movement of small particles or cells is 10⁴ -10⁵per second. A flow cytometer may be used to practice the presentinvention, if two conditions are met: (1) the transponders are smallenough to pass through the flow chamber, and (2) the design of the flowchamber of the flow cytometer is modified to include an antenna andscanner for collecting the electromagnetic radiation emitted bytransponders.

A miniature transponder is depicted in FIGS. 5 and 6. The source of theelectrical power for the transponder 12a is at least one photovoltaiccell 40 within the transponder 12a, illuminated by light, preferablyfrom a laser (not shown). The same light beam induces the fluorescenceof fluorogenic molecules immobilized on the surface of the transponder12a. The transponder 12a includes a memory element 42 that may be of theEEPROM type. The contents of the memory is converted from the digitalform to the analog form by a Digital-to-Analog converter 44 mounted onthe transponder 12a. The signal is amplified by an amplifier 45, mixedwith the carrier signal produced by an oscillator 48, and conducted tothe outside of the transponder 12a by an antenna 50.

The contents of memory of the miniature transponder can be permanentlyencoded, e.g., as ROM memory, during the manufacturing process of thetransponder, different batches of transponders being differentlyencoded. Preferably, the memory of the transponder is user-programmable,and is encoded by the user just before, during, or just after thebiological material is deposited on the surface of the transponder. Auser-programmable transponder 12a must have the "write" feature enabledby the antenna 50, amplifier 44 and the Analog-to-Digital converter 46manufactured on the transponder 12a, as well as the dedicatedscanner/write device 27.

In a preferred embodiment, the signal from the scanner is transmitted bymodulating the intensity of the light illuminating the transponder 12a,which also actuates the photovoltaic cell power source 40.

The advantages of the miniature transponder of FIGS. 5 and 6 areseveral-fold. First, the transponder dimensions are reduced relative toa conventional transponder, because most of the volume of a conventionaltransponder is occupied by the solenoid. The current design will enablethe production of cubic transponders on the order of 0.01 to 1.0 mm, asmeasured along a side of the cube, and preferably 0.05 to 0.2 mm.

Second, a large number of transponders can be manufactured on a singlesilicon wafer. As depicted schematically in FIG. 6, a silicon wafer 60is simply cut to yield active transponders 12a. Third, the transponder,according the new design, will not need the glass capsule as anenclosure, further reducing the size of the transponder. Siliconedioxide (SiO₂) would constitute a significant portion of the surface ofthe transponder, and SiO₂ has chemical properties like glass that allowderivatization or immobilization of biomolecules. Alternatively,microtransponders may be coated with a variety of materials, includingplastic, latex, and the like.

Finally, most importantly, the narrow focus of the beam of the laserlight would enable only one transponder to be active at a time duringdecoding, significantly reducing noise level. Advanceduser-programmability is desirable as well and, preferably, variousmemory registers are addressable independently, i.e., writing in oneregister does not erase the contents of other registers.

FIG. 7 shows the analytical instrumentation and transport system used inan embodiment of the present invention. A quartz tube 20 is mounted inthe readout window 22 of a fluorometer 24. The quartz tube 20 isconnected to a metal funnel 26. The length of the quartz tube 20 issimilar to the dimensions of the transponder 12. Transponders 12 are fedinto the metal funnel 26, and pass from the funnel 26 into the quartztube 20, where the fluorescence is read by the fluorometer 24 and thetransponder 12 is decoded by the scanner 27, and then exit through ametal tube 28 and are conducted to a collection vessel (not shown). Themetal funnel 26 and metal tube 28 are made of metal shield transponders12 outside of the read window 22 by shielding from the electromagneticsignal from the scanner 27. This shielding prevents the scanner signalfrom reaching more than one transponder 12, causing multipletransponders 12 to be decoded.

Minimal modification of the fluorometer 24 would be needed in thevicinity of the location that the tube occupies at the readout moment toallow for positioning of the transponder reading device. To assurecompatibility with existing assays, the glass surrounding thetransponder could be coated or replaced with the type of plasticcurrently used to manufacture beads.

In a preferred design, depicted in FIG. 8, a modified flow cytometer isused with the miniature transponder of the present invention. A metalcoil antenna 30 is wrapped around the flow cell 32 of a flow cytometer29. The transponders 12a pass through the flow cell 32, and are decodedby the scanner device 27. The signal carrying the data sent from thetransponders 12 is amplified by an amplifier 34 and processed by thescanning device 27. As the transponders 12a are decoded, fluorescencefrom the transponders 12a is detected and analyzed by the flow cytometer29.

The examples below illustrate various aspects of this invention.

EXAMPLE 1 PREPARATION OF DERIVATIZED GLASS BEADS HAVING TRANSPONDERS

The outside glass surface of transponders (e.g., manufactured by BMDS)is derivatized in the following process.

1. Aminoalkylsilane treatment

First, the transponders are cleaned by washing with xylene, followed bya 70% ethanol rinse and air drying. Then, the transponders are submergedfor about 30 seconds in a 2% solution of aminopropyltriethoxysilane(Cat.#A3648, Sigma, St. Louis, Mo.) in dry acetone. The glass beads arethen sequentially rinsed with dry acetone and distilled water, and thenair dried. This procedure is described in Pierce catalog (pp. T314-T315of the 1994 catalog, Pierce, Rockford, Ill.).

2. Attachment Of A Linker To Aminoalkylsilane-Treated Glass

The aminoalkylsilane-treated transponders are immersed in a 10 mMsolution of a homobifunctional NHS-ester cross-linker, BS³,bis(sulfosuccinimidyl)suberate (Pierce Cat. #21579, described on p. T159of the 1994 Pierce catalog) in 100 mM phosphate buffer (pH 7.0-7.4) for5 to 60 minutes at room temperature. The exact incubation time isoptimized for each treatment. The transponders are then rinsed withwater, submerged in a 10-100 μM protein solution in 100 mM phosphatebuffer (pH 7.4-8.0), and incubated at room temperature for 2-3 hours.The transponders are rinsed three times with 100 mM phosphate buffer (pH7.4-8.0). The unreacted sites on the glass are blocked by incubating inBlocker BLOTTO in phosphate-buffered saline (PBS, Pierce, Cat. #37526)for 2 hrs. The transponders are rinsed three times with 100 mM phosphatebuffer (pH 7.4-8.0), and stored in this buffer at 4° C.

The described procedure, found in Enzyme Immunodiagnostics, E. Kurstak,Academic Press, New York, 1986, pp. 13-22, works with many proteins.However, since properties of proteins can differ widely, for someproteins alternative immobilization schemes may have to be used.

EXAMPLE 2 SINGLE ASSAY FOR A PROTEIN ANALYTE

The purpose of this assay is to obtain a qualitative indication of thepresence of human chorionic gonadotropin (hCG) in the sample, which inthis example is a solution of hCG labeled with fluorescein in PBSbuffer. Another purpose is to be able to retrieve during the course ofthe assay the identification of the source of hCG used to prepare thesample.

The surface of the transponder (model IPTT-100, manufactured by BMDS) isderivatized as in Example 1 by aminoalkylsilane treatment, and the BS³linker is attached to the aminoalkylsilane treated glass. A monoclonalantibody raised against hCG is then conjugated to the linker to form anembodiment of the solid phase particle of the present invention.

A transponder derivatized with the anti-hCG antibody is immersed in 1 mlof PBS in a test tube. A fluorescein-labeled HCG preparation is added tothe test tube. The final concentration is between 50 pg/ml and 50 μg/ml.The transponder is incubated at room temperature for 30 minutes. Duringthat time, six alphanumeric characters constituting the samplelot-number identifying the source of hCG is encoded into the memory ofthe transponder using a dedicated read/write scanner. Additionalinformation such as the lot number of the antibody preparation used, orthe name of the patient who donated the serum containing hCG may also beencoded on the transponder. After a series of extensive washes over aperiod of 5 minutes, the transponder is placed in 1 ml of fresh PBSbuffer. The transponder is then placed in a fluorometer, and thefluorescence intensity (FI) is measured and recorded. The FI readout isnormalized with respect to positive control transponders which wereexposed to fluoresceinated bovine serum albumin instead offluoresceinated hCG. The electronic memory of the transponder is decodedusing a dedicated scanner to obtain the lot number of the hCGpreparation.

The advantage of using the transponder in this example instead of aprior art solid phase particle is that there is no need to maintain anassociation between the test tube and the solid phase at all timesduring the assay. Instead, after electronically recording the lotnumber, the transponder can be separated from the original containerwithout losing track of the lot number of the sample to which it wasexposed. The transponder can be mixed with other transponders exposed toanalytes having different lot numbers without losing information aboutthe presence of hCG in the analyte.

EXAMPLE 3 ELECTRONICALLY INDEXED SOLID PHASE ASSAY FOR HUMAN IgAl ANDIgGl

A total of eight transponders (e.g., BMDS model IPTT-100) arederivatized according to the procedure of Example 1, with twoantibodies, human IgAl (Cat. #I2636) and IgGl (Cat. #I4014) obtainedfrom Sigma (St. Louis, Mo.). Thus, two groups of transponders carryingthese two antibodies are obtained. Four transponders of each of the twogroups of transponders are encoded with the index numbers A1, A2, A3 andA4, and G1, G2, G3 and G4, respectively, by the read/write scannerdevice (BMDS). The letter corresponds to the type of immunoglobulin usedto derivatize the transponder, and the digit gives the tube number.Transponders are distributed into assay tubes, each tube containing onetransponder of each type. Thus, tube 1 contains transponders encoded A1and G1, tube 2--A2 and G2, etc.

The following set of analytes is prepared at a concentration between 50pg/ml and 50 μg/ml in PBS:

Analyte 1: Mixture of monoclonal antibody to human IgAl labeled withFITC (Cat. #F6016), and monoclonal antibody to human IgG1 labeled withFITC (Cat. #F5016).

Analyte 2: Monoclonal antibody to human IgAl labeled with FITC.

Analyte 3: Monoclonal antibody to human IgGl labeled with FITC.

Analyte 4: No antibody present.

2 mls of analyte 1 is added to tube 1, containing two transponders, oneof each group as described above. Similarly, 2 mls of analyte 2, 3 and 4are added to tubes 2, 3 and 4, respectively, also containing twotransponders each. The tubes are kept at room temperature for 30minutes, after which the transponders are washed three times with 5 mlPBS buffer. The fluorescence of each of the transponders is quantitatedby using a FluorImager (Molecular Dynamics), and the encoding of eachtransponder is determined by using the read/write device (BMDS).

The assay described is direct, since the concentration ofFITC-derivatized antibody is measured. Alternatively, the assay can beconfigured in a competitive format to measure the concentration ofunderivatized antibodies, such as those present in sera. Moreover, thenumber of analytes that can be tested in one tube is limited only by therequirement that the total volume of the transponders needed in thesingle tube should not be much larger than the sample volume.

EXAMPLE 4 MULTIPLEX ASSAY FOR ANTIBODIES EMPLOYING PEPTIDES IMMOBILIZEDON TRANSPONDERS

Peptides can be immobilized on the surface of the transponders' glassenvelope by either chemical synthesis or conjugation. The glass surfaceof the transponder (e.g., AVID) is first derivatized withaminopropyltriethoxysilane, creating a suitable solid support forchemical peptide synthesis. The amino groups of the alkyl chainsattached to the support are appropriate for initiating peptide synthesisby forming the amide bond with the C-terminal residue of the peptidewhen standard Fmoc or Boc chemistries are used. The resulting peptidecan be deprotected according to standard protocols without cleaving thepeptide from the support.

Alternatively, peptides previously synthesized or isolated can beattached to the treated glass surface using the cross-linker andprotocol of Example 1. The requirement will be the presence of a primaryamine group in the peptide (such as N-terminal amine), or a secondaryamine group. The assay configuration is identical to Example 1, exceptthat the proteins (i.e. monoclonal antibodies) of Example 1 are replacedwith peptides in this Example.

EXAMPLE 5 A DIAGNOSTIC KIT FOR PERFORMING AN ELECTRONICALLY INDEXEDMULTIPLEX ASSAY FOR THE HEPATITIS C VIRUS

The kit is used to simultaneously determine the presence of antibodiesto four Hepatitis C Virus (HCV) antigens in human serum or plasma, or inmixtures of purified anti-HCV antibodies prepared in the laboratory. TheHCV antigens are as follows: (1) core, (2) NS3, (3) NS4, N-terminalpart, (4) NS4, C-terminal part.

The constituents of the HCV reagent kit are as follows:

1. Reagent A, Specimen Diluent, 10 Mm Tris-HCL, pH 7.5. Preservative:0.1% sodium azide.

2. Reagent B, Probe. Goat antibody to human IgG (H+L), conjugated tobiotin. Minimum concentration; 0.1 pg/ml. Preservative: 0.1% sodiumazide.

3. Reagent C, Conjugate. Rabbit antibody to biotin, conjugated toalkaline phosphatase. Minimum concentration: 0.1 pg/ml. Preservative:0.1% sodium azide.

4. Reagent D, Chromogen. 5-bromo-4-chloro-3 indolyl phosphate (0.1%).Preservative: 0.1% sodium azide.

5. 20 Test Vessels. Each vessel is a 2 ml test tube and contains 4transponders conjugated to four HCV antigens. The antigens are appliedat a minimum of 1 ng per transponder. The transponders areelectronically encoded with numbers 1,2,3 and 4, corresponding toantigens (1),(2),(3) and (4) respectively.

6. 1 Vial (0.1 ml) Accessory Positive Control. It is an inactivatedhuman plasma containing antibody to HCV, non-reactive for HBsAg andantibody to HIV-1/HIV-2. Minimum titer: 1:2. Preservative: 0.1% sodiumazide.

7. 1 Vial (0.1 ml) Accessory Negative Control. It is human plasmanonreactive by FDA licensed tests for antibody to HCV, and non-reactivefor HBsAg and antibody to HIV-1/HIV-2. Preservative: 0.1% sodium azide.

8. Wash buffer. 10 mM Tris-HCL, pH 7.5.

9. Enzyme Reaction Buffer, 100 mM Tris-HCL, pH 8.0.

10. Bar coded calibration data sheet.

In an alternative configuration of the kit, the chromogen, reagent 4above, is replaced with a fluorogen, item 4a and Reagent 4a, namely:

4a. Reagent 4-a, Fluorogen, precipitating substrate for alkalinephosphatase (0.1%). The substrate is attophos reagent, manufactured byJBL Scientific, San Luis Obispo, Calif. Preservative: 0.1% sodium azide.

The procedure for performing the assay on a single sample of unknowncomposition with regard to HCV antibodies is as follows. Three testvessels, X, Y and Z are placed in a rack. Sample is added to vessel X,Accessory Positive Control added to vessel Y, Accessory Negative Controladded to vessel Z. Appropriate amounts are determined for each lot ofreagents, but approximate volumes are 10-100 μl sample or controlsdiluted with the Wash Buffer to the final volume of 2 ml. The sample andbuffer are thoroughly mixed, and incubated for 30 minutes at roomtemperature, after which the transponders in the vessel are washedextensively for 5 minutes with the Wash Buffer. Reagent B is then added,and the vessel is incubated for 30 minutes, after which the transpondersare washed. Reagent C is then added, and the vessel is incubated for 30minutes, after which the transponders are washed. One ml of the enzymereaction buffer is then added to the vessels, followed by 1 ml of thesubstrate (item 4 or 4a). The contents of the vessels is mixedthoroughly. The vessels are incubated at room temperature for 2 to 30minutes, depending on the desired sensitivity of the assay, after whichthe transponders are rinsed with the Wash Buffer to remove excesssubstrate and that fraction of the product of the reaction which did notprecipitate. The color of the transponders is then determined in aphotodiode spectrophotometer configured to measure the reflected light,or the fluorescence of the transponders is measured in a fluorometer,depending on the label used. Each optical measurement is followed by thedecoding of the electronic memory of the transponder and associated withthe optical measurement.

I claim:
 1. A solid phase for use in assays for biomolecules,comprising:(a) a particle and a transponder, wherein the transponder islocated inside said particle or is attached to the surface of saidparticle, the transponder comprising a radio transmitter-receiverencoded with data, the data is transmitted to a receiver in response toa specific dectromagnetic signal; and (b) a member of a biomolecularbinding pair attached to the surface of the pattide, wherein thebiomolecule specifically binds to the biomolecular binding pair member.2. The particle of claim 1, wherein the surface of the particle isglass, latex or plastic.
 3. The particle of claim 1, wherein thebiomolecular binding pair is an antigen-antibody pair.
 4. A method ofdetecting a member ofa biomolecular binding pair in a sample, comprisingthe steps of:(a) providing at least one solid phase particle, saidparticles having a transponder located inside or attached to each ofsaid particles, the transponders having memory dement and an indexnumber encoded on the memory elements creating at least one class oftransponders, each class having a different index number; (b) the solidparticles having a first member of said biomolecular binding pairattached to a surface thereof; (c) contacting the solid phase particleswith a sample to cause a second member of the biomolecular binding pairto specifically bind to the first member attached to the solid phaseparticle; (d) adding a label reagent to the sample, said label reagentspecifically binds the second member of the biomolecular binding pair onthe solid phase particle, and analyzing the solid phase particles todetect the presence of the label indicative of binding of the secondmember present in the sample to said first member; and (e) decoding theindex number encoded on the transponders using a scanner device toidentify the class of the transponders to which the second member of thebiomolecular binding pair is bound.
 5. The method of claim 4, whereinthe index number is encoded on the transponder memory element by thetransponder manufacturer.
 6. The method of claim 4, wherein the indexnumber is encoded on the transponder memory element by the user with ascanner device.
 7. The method of claim 4 wherein the label is achromophore.
 8. The method of claim 4 wherein the label is afluorophore.
 9. The method of claim 4, wherein the label is achemiluminescent agent or a bioluminescent agent.
 10. The method ofclaim 4, wherein an outer surface of the transponders is glass, plasticor latex.
 11. The method of claim 4 wherein the index number comprisesphysical or chemical characteristics of the biomolecular binding pairmember deposited on the solid phase.
 12. The method of claim 4 whereinthe index number comprises identifying characteristics of the sample.13. A method of detecting at least two biomolecules in a sample, saidbiomolecules being a second member of a biomolecular binding pair,comprising the steps of:(a) introducing into the sample at least twopopulations of solid phase particle, said particles having a transponderlocated inside or attached to each of said particles, each population ofparticle having a first member of a biomolecular binding pair attachedto its surface, and the transponders in the first population beingencoded with a different index number than the transponders of thesecond population, wherein, the first member on each population ofparticles specifically binds one of said second members present in saidsample; (b) contacting the solid phase with a label reagent thatspecifically binds to the second member of the biomolecular bindingpair, and analyzing the particles to detect a label indicating bindingof the second biomolecular binding pair member to the first biomolecularbinding pair member; and (c) decoding at least a portion of thetransponders to determine the populations of the transponders to whichthe second member is bound.
 14. The method of claim 13, wherein saidbiomolecules comprise nucleic acid sequences and the solid phaseparticles comprise at least three populations of solid phase particles,each particle comprising a transponder and each particle having anoligonucleotide attached to a surface of the particle, each of the threepopulations having a different oligonucleotide sequence attached to theparticle and each of the populations of particles being encoded with adifferent index number.
 15. A method of performing a multiplex solidphase assay for biomolecules in a sample, said biomolecules being asecond member of a biomolecular binding pair comprising the steps of:(a)providing multiple solid phases comprising particles, said particleshaving a transponder located inside or attached to each of saidparticles, the transponders having memory elements encoded with an indexnumber creating two or more classes of transponders, each class having adifferent index number, each class of solid phase particles having afirst biomolecular binding member immobilized on a surface thereof,wherein, the first member on each class of particles specifically bindsone of said second members present ill said sample: (b) contacting thesolid phase particles with a sample to cause two or more differentsecond members of a biomolecular binding pair in the sample to bind oneof said first biomolecular binding pair member on the particles; (c)contacting the solid phase particles with label reagents thatspecifically bind to the second member of the biomolecular binding pair;(d) washing the solid phase particles to remove unbound samplecomponents; (e) analyzing the solid phase particles to detect the label,which indicates binding of the second biomolecular binding pair memberto the first biomolecular binding pair member; and (f) decoding theindex number encoded on at least a portion of the transponders toidentify the class of transponders to which a biomolecule is bound. 16.A kit for detecting the presence of a member of a biomolecular bindingpair in a sample, comprising:(a) at least one assay vessel, containingat least one solid phase particle, the particle comprising atransponder, the transponder having a memory element and the particlehaving a primary layer of biomolecules bound to a surface of theparticle; (b) at least one probe reagent, comprising one member of thebiomolecular binding pair; and (c) at least one label reagent that bindsselectively to the probe reagent.
 17. The kit of claim 16, furthercomprising:(a) at least one positive control, comprising a solution of amember the biomolecular binding pair; and (b) at least one negativecontrol, comprising a solution free of the biomolecular binding pairmember.
 18. The kit of claim 17, further comprising a sample diluentbuffer solution.
 19. The kit of claim 16, wherein the primary layercomprises protein antigens.
 20. The kit of claim 16, wherein the primarylayer of biomolecules comprise vital antigens.
 21. The kit of claim 16,wherein the biomolecular binding pair member to be detected comprises acell.
 22. A method of detecting a member of a biomolecular binding pairin a sample comprising the steps of:(a) providing at least one solidphase particle, said particles having a transponder located inside orattached to each of said particles, the transponders having memoryelements and an index number encoded on the memory elements creating atleast one class of transponders, each class having a different indexnumber; (b) the solid particles having a first member of saidbiomolecular binding pair attached to the surface of the solid phaseparticles; (c) contacting the solid phase particles with a sample and alabel reagent to cause a second member of the biomolecular binding pairpresent in said sample, and the label reagent to competitively bind tothe first member attached to the solid phase particle; (d) analyzing anddetecting the presence of the label on the solid phase particle as anindirect determination of the presence of the second member in saidsample; (e) decoding the index number encoded on the transponders usinga scanner device to identify the class of transponders to which thesecond member of the biomolecular binding pair is bound.
 23. A method ofdetecting at least two biomolecules in a sample, said biomolecules beinga second member of a biomolecular binding pair, comprising the stepsof:(a) providing at least two populations of solid phases comprisingparticles, said particles having a transponder located inside orattached to each of said particles, each population of particle having afirst member of a biomolecular binding pair attached to its surface, andthe transponders in the first population being encoded with a differentindex number than the transponders of the second population, wherein,the second members present in said sample competitively binds one ofsaid first member on each population of the solid phase particles; (b)contacting the solid phase particles with a sample and label reagents tocause the second members present in said sample, and the label reagentsto competitively bind to one of said first member on the solid phaseparticles; (c) analyzing the particles to detect a label to indirectlydetermine the presence of the second biomolecular binding pair member inthe sample; (d) decoding at least a portion of the transponders todetermine the populations of the transponders.
 24. A method ofperforming a multiplex solid phase assay for biomolecules in a sample,said biomolecules being a second member of a biomolecular binding pair,comprising the steps of:(a) providing multiple solid phases comprisingparticles, said particles having a transponder located inside orattached to each of said particles, the transponders having memoryelements encoded with an index number creating two or more classes oftransponders, each class having a different index number, each class ofsolid phase particles having a first biomolecular binding memberimmobilized on a surface thereof, wherein, the second members present insaid sample competitively bind to one of said first member on themultiple solid phase particles; (b) contacting the solid phase particlewith a sample and label reagents to cause the second members and thelabel reagents to competitively bind to one of said first member on themultiple solid phase particles; (d) washing the solid phase to removeunbound sample components; (e) analyzing the solid phase to detect thelabel, which indirectly indicates binding of the second biomolecularbinding pair member to the first biomolecular binding pair member; and(f) decoding the index number encoded on at least a portion of thetransponders to identify the class of transponders to which abiomolecule is bound.